S. pombe, despite being a eukaryote, has been studied extensively for its high versatility in genetics, molecular biology and cellular biology as a unicellular organism (Nasim A. et al. eds., Molecular biology of the fission yeast, Academic Press, 1989). In its cultures, monosaccharides such as glucose and fructose are used as the main carbon sources. It is known that in a culture medium lacking these monosaccharides, expression of invertase, the enzyme that degrades sucrose into glucose and fructose, is induced to secure the carbon source necessary for its growth (Moreno S. et al., Arch Microbial. 142, 370, 1985).
S. pombe invertase is and is a high-molecular weight glycoprotein located on the cell surface with a molecular weight of about 205000, 67% of which is attributed to sugar chains composed of equimolar amounts of mannose and galactose residues. Molecular weight and amino acid studies of the protein moiety of the pure enzyme and experiments using antibodies have shown high similarlity between S. pombe invertase and the invertase from the budding yeast Saccharomyces cerevisiae from the viewpoint of protein chemistry (Moreno S. et al., Biochem. J. 267, 697, 1990). It is also known that a drop in glucose concentration de-represses synthesis of invertase (Mitchinson J. et al., Cell Sci 5, 373, 1969).
Induced invertase synthesis (de-repression) is also observed in Saccharomyces cerevisiae. Previous detailed studies on genetic regulation of invertase expression, the biosynthetic pathway and the structure of the sugar chain moiety have shown that Saccharomyces cerevisiae invertase is encoded by six overlapping genes, SUC1 to SUC5 and SUC7, on one chromosome and that activation of at least one of these SUC genes leads to utilization of sucrose and raffinose (Hohmann S. et al., Curr Genet 11, 217, 1986).
In contrast, with respect to S. pombe, although purification of the invertase protein has been reported (Moreno S. et al., 1985), no invertase genes had been identified until the present inventors and coworkers recently reported two overlapping invertase genes inv0.sup.+ and inv1.sup.+ in S. pombe. Because inv0.sup.+ is likely a pseudogene having an incomplete open reading frame, inv1.sup.+ is the only one gene encoding S. pombe invertase, which is supposed to confer the ability to grow on sucrose even in the absence of other carbon sources ("Kobogaku" edited by Yositaka Hashitani, Iwanami Shoten, 1967).
Analysis of the promoter region of the isolated gene suggested that a specific sequence between the 1st and 62nd base pairs is involved catabolite repression.
In Saccharomyces cerevisiae, the SUC2 gene is transcribed into two messenger RNAs (mRNAs) from different transcription initiation sites. The shorter one is a constitutive mRNA encoding the intracellular invertase, while the longer one is a mRNA encoding the catabolite-repressible secretory invertase with a de-repression ratio of not less than 200 (Carlson M. et al., Mol. Cell. Biol. 3, 439, 1983). Analysis of the promoter region for the longer mRNA suggested that the transcription initiation factor binds to a specific repeated sequence between positions -650 and -418 (Salokin L et al., Mol. Cell. Biol. 6, 2314, 1986). The region between positions -418 and -140 has been shown to be necessary for glucose repression.
These regions in the SUC2 gene showed no significant homology with the inv1.sup.+ upstream region between positions 1 and 2809. However, multiple copies of a so-called 7-bp motif with the sequence (A/C)(A/G)GAAAT, which is repeated at five sites in the region indispensable for glucose derepression, have been found in the inv1.sup.+ upstream region. Further, while palindrome stem-loops have been identified at almost the same positions in the upstream regions of glucose-repressible genes (SUC, MAL and GAL), palindrome sequences have also been found in the upstream region of the inv1.sup.+ gene from S. pombe. These sequences are anticipated to play an important role in glucose repression in S. pombe.
The yeast S. pombe is phylogenetically different from Saccharomyces cerevisiae. It is quite different from other yeasts in the chromosome structure and various mechanisms for genome replication, RNA splicing, transcription and posttranslational modification, and rather resembles animal cells in some of these aspects. For this reason, S. pombe is widely used as a eukaryotic model (Giga-Hama and Kumagai, eds., Foreign gene expression in fission yeast Schizosaccharomyces pombe, Springer-Verlag, 1997).
S. pombe is also widely used as a host for expression of heterologous protein genes and known to be suited especially for expression of genes from animals including human (JP-A-5-15380 and JP-A-7-163373). For its advanced membrane structures including the Goldi body and the endoplasmic reticulum, S. pombe is also used for expression of membrane proteins and shows high level expression. For S. pombe, constitutive expression vectors (pEVP11, pART1 and pTL2M) and an inducible expression vector using the promoter region of the nmt1.sup.+ gene (pREP1) are usually used as expression vectors. No S. pombe expression vectors of the GAL type or the SUC type have been known though these types of vectors are commonly used for Saccharomyces cerevisiae.
The expression of the SUC2 gene from Saccharomyces cerevisiae in S. pombe has been shown to be constitutive, not catabolite repressible, though the expression product contains galactose residues conferred by the host (Zarate, V. et al., J Applied Bacteriology, 80, 45, 1996), suggesting differences between S. pombe and Saccharomyces cerevisiae in the mechanism for catabolite repression of invertase. The differences are of great significance because the promoter from Saccharomyces cerevisiae usually used by those skilled in the art for construction of inducible expression vectors of the invertase type (the SUC2 type) is not applicable to S. pombe vectors. Therefore, development of S. pombe vectors of this type has been long delayed.
On the other hand, the present inventors constructed an expression vector using the secretion signal gene encoding the secretion signal in the precursor of a S. pombe mating pheromone (WO96/23890). However, this secretion signal gene is not an all-purpose secretion signal gene, and other secretion signal genes that function in S. pombe are desired for production of some types of protein.